Effect Of Aerobic Exercise And Rosemary Extract On Learning And Memory In Aged And Young Rats

In a live organism, although, aging may also refer to the positive processes of maturation or acquisition of a desirable quality, but usually refers to a sequence of time-dependent changes that decrease physiological and functional capacity. Aging process is often accompanied by cognitive and memory decline and an increased chance, involved in neurodegenerative disorders (Hung, Chen et al. 2010),(Mattson and Magnus 2006). Recently, physiological, biochemical and structural changes, known in aging process may provide a basis for further research line in order to slowing down the brain aging and prevention of neuronal loss or damage. During the metabolism processes in the cell, concentration of free radicals during normal oxygen metabolism may be restricted physiologically by various antioxidants cell source and a balance exists between provident and antioxidant processes (Chen, Lin et al. 2008). A primary place for the production of free radicals or ROS is the mitochondrial electron transport chain (ETC). During oxidative phosphorylation, electrons can flee ETC between the respiratory complexes and combine with oxygen to create the superoxide anion radical. Superoxide can react with different cellular components directly (Harman 1972) . So if the cell function was declined in age-dependent manner, accumulation of molecular oxidative damage may occur (Sharman, Bondy et al. 2007).
Among the herbals reported to have antioxidant activity, rosemary (Rosmarinus officinalis L.) is one of the most widely commercialized plant, used as a culinary herb for flavoring and as an antioxidant in processed foods (Zheng and Wang 2001). Carnosic acid, the most abundant antioxidant found in the leaves of rosemary, is a lipophilic antioxidant that scavenges singlet oxygen, hydroxyl radicals, and lipid peroxyl radicals. So, it can prevent the lipid peroxidation and disruption of biological membranes (Munn??-Bosch and Alegre 2001).
Studies have shown that aerobic exercise has benefit e'ects on brain function (van Praag 2008) and also cardiovascular, pulmonary, and the musculoskeletal systems (Bernardi, Tramontina et al. 2013).
It is demonstrated that moderate physical activity improves memory and learning by affecting on the hippocampus (Bernardi, Tramontina et al. 2013). Hippocampus plays important roles in general cognition, mood regulation, response to stress, learning, and memory and even in encoding predictions for future events. (Balu and Lucki 2009)
There are some tasks to evaluate learning and memory functions. The Morris water navigation task, also known as the Morris water maze, is a behavioral procedure widely used to study spatial learning and memory. (Morris, Garrud et al. 1982)
The Passive avoidance learning, using shuttle box apparatus, is believed to be based on contextual memory, which is associated with the place and the event of "being given the electric shock in the dark box". (Hirsh 1974)
The aim of this study was to evaluate the effect of aerobic exercise and Rosemary extract on learning and memory in aged and young rats.
2. Materials and methods
2.1. Animals:
The male Wistar rats (animal lab of Iran University of Medical Sciences, Tehran, Iran) (50: aged, 18 -month old and 50: young, 2 weeks old) were used in this study, housed in the animal lab in laboratory cages (5 animals/cage) under a 12 h light/dark cycle. The room temperature maintained at 21?? 2 ??C and they had free access to food and water. Animals were trained during the light cycle and their experiments were carried out according to the ethical committee for the use and care of laboratory animals of Iran University of Medical Sciences. All efforts made to minimize animal suffering.
Animals were divided in ten groups: control groups (IY: Intact young, IO: Intact Old); vehicle groups (VY: vehicle Young, VO: vehicle Old); exercise groups (EY: Exercise Young, EO: Exercise Old); rosemary extract groups (RY: Rosemary Young, RO: Rosemary Old); and exercise & rosemary extract groups (ERY: Exercise & Rosemary Young, ERO: Exercise & Rosemary Old). Animals in the intact groups have been remained in their home cages throughout the experiment. Animals in the vehicle groups have been spent 10 minutes on the turned off treadmill and received orally 1 ml of distilled water daily, using animal-feeding incubation needles (Perfecta, German). Animals in rosemary groups received daily, rosemary extract (100 mg/kg) dissolved in 1 ml distilled water. Animals in exercise groups had been administered daily, aerobic exercise (by treadmill apparatus) these procedures were performed for 12 weeks.

2.2. Treadmill exercise:
Rats in exercise groups have got familiar to run on a motor-driven leveled treadmill (Pishroo andisheah sanate A1400Y10, Iran) at a low speed 10 m/min each day during 7 days. Then, the animals started to enter in a programmed exercise (60 min/d, 0% slope) for 12 weeks. The training speeds began at 12 m/min and reached to 22 m/min by the end of the experiment.

2.3. Rosemary Extract:
Rosemary Extract ( 40% carnosic acid) was brought from human Geneham biomedical technological company (china) (RAP20-110401).

2.4. Behavioral tests:

2.4.1 .Morris water maze performance:

The Morris water maze (MWM) evaluates the spatial learning and memory in rats. In this study, we used the protocol described by with a little changes (Morris 1984). The tank, we applied for MWM was 210 cm in diameter, 50 cm high sidewalls, made of stainless steel and painted black color. The tank filled with water with (21 ?? 1 ??C) for young groups and (25 ?? 1 ??C) for old groups, to a depth of 25 cm. There was furniture in the room that provided visual cues and several posters mounted walls of the room. During this experiment, the room was set with indirect incandescent illumination. The tank divided geographically manner into four quadrants North East (NE), North West (NW), South East (SE), and South West (SW). A round escape platform (diameter: 10 cm) was located 2 cm beneath the surface of the water in the center of one of the four quadrants of the pool. The position of the escape platform remained consistent for all animals across acquisition phase test. An infrared video camera (Panasonic, WV-BP330/GE, Japan) mounted directly above the water maze tank for record the swim path of the animals. All tests began at 11 a.m. until 1 p.m. Each animal received one block of four trials per five consecutive days. In each trail, we placed the animal in the water, facing the wall of the tank at one of the four designated starting points for swimming and finding the hidden platform. The hidden platform was hidden in the NE quadrant of the maze. Starting points varied in randomly fashion. Therefore, in each trial the subject started from one of the four quadrants, and never started from the same place on any two consecutive trials. During each trial, the animal allowed to have a time of 60s to find the hidden platform. If the animal found the platform, she could remain on it for a 30s additional time. On the other hand, if the rat failed to find the platform within 60s, observer placed her on the platform for 30s later. During the first 5 days, the platform position remained constant. On 6th day (probe test), the platform was removed from the maze. A computerized video tracking system Ethovision XT version 5 (Leesburge, VA, USA) recorded the data in this test.
2.4.2. Passive avoidance (PA) task:
The passive avoidance task performed using the shuttle box to study the short-term memory in rats. The shuttle box consists of two equally sized compartments, with a sliding door between two compartments and a grid floor for the delivery of an electric foot shock. An electric light bulb illuminates one compartment, while the other compartment remains in the dark. In the training session, each animal was placed in the light chamber, facing away from the sliding door. When the animal entered to the dark compartment, the sliding door was quietly lowered and a 0.5 mA foot shock was delivered for 2 seconds through the grid floor. During the test session, the animal was placed in the light compartment once more to enter the dark compartment, but the foot shock was absent. The latency to step through was documented by a blind observer (Silva, Felicio et al. 1999). The time of latency to the dark chamber was recorded up to a maximum of 300 seconds (LTP day 2).

3. Statistical analysis
Data were expressed as means ?? standard error of the mean (S.E.M.) and results analyzed by SPSS statistical software package (version 16). One-way ANOVA and Tukey's post-hoc comparison were performed and two-way repeated measurements tests. Data in passive avoidance learning test were analyzed by paired sample t-test. P'''0.05 was considered as significant.

4. Results:
4.1. Morris Water Maze Test
4.1.1. Traveled Distance:
Traveled distances to find the platform is one of the parameters evaluated in the water maze, which defines the learning process. In the training phase of the Morris water maze test analysis two way ANOVA, respectively for groups, days and groups-days F(9,27) = 33.73, F(4,32)=39.63, F(36,35)=4.32 ( p < 0.05).
Results in aged rats showed that animals in EO, RO and ERO groups traversed signi'cantly shorter path lengths to 'nd the hidden platform compared to the animals in IO and VO groups (P<0. 01).
Results in young rats showed that animals in EY group traversed signi'cantly shorter path lengths to 'nd the hidden platform compared to the animals in IY group (P <0.05) (Figure 1).

Figure 1: Comparison of the means of traveled distance for all groups in learning phase. The traveled path lengths in EO, RO and ERO groups were signi'cantly shorter to 'nd the hidden platform compared to IO and VO groups of animals( ** P<0.01). The traveled path lengths in EY young rats exhibited signi'cantly shorter compared to IY group of animals (*P<0.05).

4.1.2. Escape Latency:
Escape latency (EL) is the amount of time to find the target platform, the other parameters examined in the water maze to assess learning. Analysis two way ANOVA ,respectively for groups, days and groups-days F(9,81) = 16.74, F(4,83)=170.82, F(36,100)=2.06 ( p < 0.001).
Results in figure 2 shows that animals in RO and ERO aged groups exhibited signi'cantly shorter latency times to 'nd the hidden platform compared to the animals in IO and VO groups (P <0.05). In addition, there is a significant difference between EO and VO groups (P <0.05). In young animals, there is no signi'cantly difference between groups.

Figure 2: Comparison of mean of Escape Latency in all groups. The animals in RO and ERO aged groups exhibited signi'cantly shorter latency times to 'nd the hidden platform compared to the IO and VO groups. Animals in EO aged group exhibited signi'cantly shorter latency compared to the VO group (*P<0.05).

4.1.3. Swimming speed:
There were no significant differences in swimming speed between all groups (data not shown).
4.1.4. Time spent to target quadrant (ETQ):
In the 6th day of MWM task (probe test) or retention day of water maze, the time spent in the target quadrant was tested.
Figure 3 shows that in old rats, there is a significant increase in the time spent in the target quadrant in ERO group of animal compare to VO group (P<0.05). Although, the scores of EO and RO showed slightly, non-significant increase compare to IO and VO groups. Results in young groups show that no significant in this score in young groups.

Figure 3: Comparison of mean of time spent in the target quadrant of all groups in probe test. In aged groups, animals in ERO group significantly spent more time in the target compare to the VO group (*P<0.05).
4.2. Passive Avoidance Learning Test:
Passive avoidance tasks (shuttle box) assays avoidance learning, working memory and fear memory. As it explained before, this test should be done in two consecutive days. Data was analyzed by paired sample t-test.
4.2.1. Step ' through latency Day 1:
Figure 4 showed no significant difference between groups in the first day.
4.2.2. Step ' through latency Day 2:
In second day, the length of stay in light chamber was evaluated. Figure 4 shows that there are significant increases in the length of stay in light chamber in EO, RO and ERO groups of animals compared to IO and VO groups (P<0. 01). There was no significant difference in this score between young groups. The results of paired sample t-test between the two consecutive days in all groups showed that there are significant increases in the scores of the first day compared to the second day in all groups (P <0. 01), exept in the scores of IO and VO groups (Figure 5).

Figure 4: Comparison of the mean of step ' through latency (shuttle box). The scores of animals in EO, RO and ERO groups in the second day show significant increases compared to the scores of IO and VO groups (**P<0. 01).

Figure 5: In all of groups, paired t-test showed significant increases in second day compared to the first day (P<0. 01), except in IO and VO groups.

5. Discussion:
The ageing process itself shows the cumulative effect of deterioration caused by free radicals in cells and tissues. Oxidative stress can damage the cell through the oxidization of cellular elements. Several studies have reported correlations between age and the accumulation of oxidative damage in cells (Head 2009; Posadas, Caz et al. 2009). In addition, the ability to perform cognitive operation, which is reduced in normal aging, seems to be results of oxidative damages in hippocampus. In general, studies show that working memory, long-term memory, and perceptual speed are reduced because of ageing (Park, Lautenschlager et al. 2002).
On the other hand, it has been reported that, running can improve some kinds of memory and learning in aged mice due to the enhanced hippocampus neurogenesis (Van Praag, Shubert et al. 2005). Rovio et al suggested that exercise improves cognitive functions and delayed the onset of Alzheimer's disease (Rovio, K??reholt et al. 2005)
Rosemary contains several antioxidant compounds and exhibits different protective effects, such as hepatoprotective, anti-hyperglycemic, anti-ulcerogenic actions (Corr??a Dias, Foglio et al. 2000) , (Sotelo-Felix, Martinez-Fong et al. 2002) hypolipidemic potential (Al-Jamal and Alqadi 2011). It has been shown that Rosmarinus officinalis extract and its antioxidant compounds inhibit lipid peroxidations and free radicals generation in vitro and in vivo (Al Sheyab, Abuharfeil et al. 2012) Carnosic acid is one of the effective antioxidant agents which is found in the leaves of rosemary (Munn??-Bosch and Alegre 2001).
In this work, we have analyzed whether learning and memory abilities in aging rats can alter due to the effects of daily 100 mg/kg rosemary extract (40% of carnosic acid) and treadmill running aerobic exercise (1hour/day and 5 days/week) in 3 months.
In this study, MWM is used to test spatial reference memory and learning by observing and recording of traveled distance, escape latency, velocity of swim, and time spent to target quadrant.
In learning phase, the results of traveled distance and escape latency illuminate that both the exercise, rosemary extracts, and also, combination of exercise - extract could be effective in reducing the traveled distanced and the time spent to find the target platform in old rat groups. Otherwise, analysis of the results in young rats shows that, exercise can decrease only the traveled distance, but no other significantly difference is seen by rosemary extract or combination between exercise ' extract in young rats.
The results in memory phase or probe test demonstrate that combination between exercise and rosemary extract could increase the time spent in target quadrate in old rats. However, there is not any significant difference in this score in young rats.
Other studies revealed that acute treadmill running increased memory de'cit (tested by passive avoidance) caused by morphine in rats (Alaei, Borjeian et al. 2006). Also, Albeck, et al. (2006) reported that forced treadmill exercise for 7 weeks decreased both the traveled distance and escape latency (in MWM test) in aged rats and there was no difference in swim speed (Albeck, Sano et al. 2006).

In addition, the passive avoidance task using the shuttle box is performed to study the short-term and working memory. Data of step ' through latency shows that both the exercise, rosemary extract and exercise ' extract combination are significantly effective in increase the length of stay in light chamber in the second day in the old rats. Also, data analysis in comporison between two consequence days shows that the time delayed to intrance to the dark room are increased in the old animals who recieved exercise, rosemary extract delivered and exercise ' extract combination in the second day compaired to the first day. These data reveals that ageing can decline learning and working memory, but exercise, rosemary extract and comporision between them can improve those scores.
Many studies have shown the health bene't of exercise (Deslandes, Moraes et al. 2009). Some evidence suggests that physical activity helps to maintain brain health and cognition and protects against the progressive cognitive decline due to the aging process (Meeusen, 2005; (Cotman, Berchtold et al. 2007). According to the literature, it seems that, in the exercise, the insulin-like growth factor 1 (IGF-1) and BDNF pathways are important targets. So, the blockage of the hippocampal receptors for IGF-1 (IGF-1R) or BDNF (TrkB) eliminate the effects of exercise on hippocampal plasticity and molecular changes, such as increases in synapsin 1, calcium/calmodulin-dependent kinase II (CaMKII), and mitogen-activated protein kinase II (MAPKII) (Vaynman, Ying et al. 2004; Ding, Vaynman et al. 2006; Kaliman, P??rrizas et al. 2011). Also, it has been suggested that lessened neurogenesis in the hippocampus is associated with cognitive de'cit (Drapeau, Mayo et al. 2003) whereas exercise enhances the neurogenesis, and therefore may contribute to cognitive functions (van Praag, Christie et al. 1999) (Van Praag, Shubert et al. 2005). In another study, it revealed that exercise by treadmill also increased neurogenesis, spatial and short-term memory scores and also suppressed the apoptosis in the dentate gyrus in the old-aged rats. This research showed that, increased effect of treadmill exercise were not observed in the young rats (Kim, Ko et al. 2010) . Chen and colleagues showed that treadmill exercise for four weeks signi'cantly, facilitated passive avoidance (PA) performance in rats (Chen, Lin et al. 2008).
On the other hand, there are some studies related to the rosemary extract and ageing. Posadas and colleagues reported that rosemary extract consumption by aged rats decreases catalase activity, lipid peroxidation and ROS levels in the cerebrum (Posadas, Caz et al. 2009). It has been revealed that Rosmarinus officinalis extract and its antioxidant compounds inhibit lipid peroxidations and free radicals production in vitro and in vivo. (Al Sheyab, Abuharfeil et al. 2012) . Also, Rasoolijazi et al. showed that carnosic acid, one of the effective compound found in rosemary extract, can ameliorate the spatial and learning memory deficits induced by the toxicity of beta-amyloid in the rat hippocampus (Rasoolijazi, Azad et al. 2013). Waggas and colleagues reported that administration of acrylamide (30mg/kg, i.p.) for one week after treatment with rosemary leaves extract (100mg/kg, i.p.) for 3 weeks caused a significant increased in epinephrine content in cerebral cortex (Waggas and Balawi 2008).
Present study shows that daily administration of 100 mg/kg rosemary extract (40% carnosic acid) and or treadmill exercise (1h/d) for 3 months could improve the memory and learning abilities evaluated by MWM and shuttle box tests in aged rats. It seems that these results are due to the effects of aerobic activity on improving the efficiency of the capillary system and trigger the plasticity in the hippocampus and antioxidant activities of rosemary extract.

6. Conclusion:
Our results demonstrated that moderate treadmill exercise and or rosemary extract caused enhanced learning) decrease traveled distance and escapes latency( in MWM and improving in learning and short memory on passive avoidance test in old rats. So, this study demonstrated that use of exercise and rosemary extract combine together could be useful in enhance learning and memory abilities in elderly.

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